| Pinctada martensii is also called Pinctada fucata, which can product the sea pearl sas one of the important economic shellfish in our country. It can be maked as the shellfish meat and product the sea pearls that south sea pearls is famous in the world.In recent years, due to the deterioration of environmental conditions as well as unscientific aquaculture breeding way leads to the degeneration of germplasm resources, the yield and quality decline of pearls, enterprise economy severely damaged, so it’s urgent need for the researchs on the genetic diversity analysis,optimized selection, genetic improvement, the breeding using molecular markers and the protection of its germplasm resources in pinctada martensii. We adopted the molecular marker technologies such as the expressed sequence tag microsatellite markers(EST-SSR), the sequence related amplified polymorphism markers( SRAP-c DNA) and the single nucleotide polymorphism(SNP) to analyzes the genetic diversity, screen the molecular markers and the SNP locis about the tyrosinase gene related with the shell colors, and measure the different expression amounts of tyrosinase gene in four shell color populations of pinctada martensii. Being aimed at providing the molecular theory basis for the identification of germplasm and the selection of shell color in pinctada martensii, which experienced the group transgenerational breeding for eight years.The main research results are as follows:1、we adopted the 10 microsatellite primers to study the genetic diversity and population structure of the four shell color populations of pinctada martensii, the results showed:(1) Forty-five alleles were detected by 10 locis, the average allele number of each loci is 4.5, the range of average allele(Na) numbers is 3.5 ~ 4.0, the range of average effective alleles(Ne) numbers is 2.288 ~ 2.730,the average of average observed heterozygosity(Ho) is 0.445 ~ 0.627, the range of average expected heterozygosity(He) is: 0.479 ~ 0.580, the range of average polymorphism information content(PIC) is 0.423~0.556 and the value size of genetic diversity of four shell color populations of pinctada martensii, in turn, is red shell color > white shell color > > black shell color> yellow shell color.(2)Three locus deviated from the hardy weinberg equilibrium highly significantly(P < 0.01), and two locus deviated from the hardy weinberg equilibrium significantly(P < 0.05) and the rest locus of four shell color populations of pinctada martensii had been with different degree of deviation. The average index of genetic differentiation(Fst) value is 1.206, the average gene flow(Nm) index is 1.8825 and the genetic differentiation is medium with no gene flow. The genetic distance(Dxy) value range is 0.1742 ~ 0.3415, the genetic similarity(Nei) ’s range is 0.7107 ~ 0.8418. Compared with F3 and F5 generation shell color selection system, the genetic diversity, genetic variation and genetic distance of four shell color populations of pinctada martensii were increased,such as the genetic structure had been changed, populations tend to be purified, and formed four new shell color strains of pinctada martensii.2、We used SRAP- c DNA technology to analyze the expression of mantle gene in four shell color populations of pinctada martensii. The results showed that: Five different bands were screened by 100 pairs of SRAP primer combinations,and eight sequences were cloned. We got 5 similar protein sequences through Blastx program comparison, one hypothetical protein, two HMP-P synthase and two Rossman folding NAD(P)(+) binding protein.The research showed that the red shell color populations of pinctada martensii have three single markers,such as the first different bands of primer 17,the second different bands of primer 17 and the different bands of primer 28. The yellow shell color population have two single markers, such as the different bands of primer 41and70. They can make as the identified molecular markers of the red and yellow color populations of pinctada martensii to provide theoretical basis for its germplasm identification.3、We used SNP molecular marker technology to screen 22 SNP mutations locus and3 missing locus in the tyrosinase gene encoding fragment between 887 bp to 1571 bp.It has 3.6 base mutations per 100 bases in the sequence. The A, C, C bases were absent in 944 bp, 945 bp and 946 bp locus. The absent sequence which could be recombined after missing. Compared with NCBI tyrosinase gene sequences in the database, the recombined sequence just lost A proline. 22 SNP locus are diallele genotype, which have six non-synonymous mutations, 16 synonymous mutations, 14 conversion and eight transversion.The results showed that:(1) The four shell color populations of pinctada martensii and the population of pinctada martensii in which the tyrosinase gene was cloned from NCBI database had significant differences, so they are not the same group.(2)Through the multiple comparison, we found that the different SNP locus in four shell color populations of pinctada martensii have significant difference in various degree. The fourteen screened SNP locus can be used as molecular markers for the identification of red shell color population. There are six identification tags of white and black shell color populations. There are three identification tags of white and yellow shell color populations. There are one identification tags of yellow and black shell color populations. These a few molecular markers can be used to the germplasm identification of our four shell color populations of pinctada martensii which were selected by our team.(3) The polymorphism(PIC) range of four shell color populations in 22 SNP locus is 0.012 ~0.012, the average is 0.2922 and 20 locus significantly deviated from the hardy weinberg equilibrium(P < 0.01). The genetic diversity is lower and the purification degree of the shell color is higher.(4) There has no significant difference in tyrosinase gene expression of four shell color populations(P > 0.05). |
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